Device and method for completing a fluidic circuit

ABSTRACT

Disclosed are assay apparatus, devices and methods that permit longitudinal capillary flow of liquid between two pieces of bibulous material which, prior to actuation, are in a non-capillary flow relationship to each other. In particular, the devices utilize a distortable member which when distorted, actuates the device by creating a capillary flow relationship between the two pieces of bibulous material. In an alternative embodiment the apparatus, devices, and methods include at least one additional piece of bibulous material.

This is a Division of application Ser. No. 07/821,338, filed Jan. 13,1992, which is a File Wrapper Continuation of application Ser. No.07/441,676, filed Nov. 27, 1989, now abandoned, the disclosures of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to assay apparatus, devices andmethods that permit longitudinal capillary flow of liquid between twopieces of bibulous material and that, prior to actuation, are in anon-capillary flow relationship to each other. In particular, the deviceutilizes a distortable member which when distorted, actuates the deviceand a capillary flow relationship is initiated between the two pieces ofbibulous material. The methods, apparatus, and devices of the presentinvention have utility in conducting assays which use capillarity totransport solutions and/or samples including assays such as enzymeimmunoassays, fluorescent immunoassays, radioimmunoassays, etc.

2. Related Art

Assay devices have been described in which liquid is transported bycapillarity through or transversely along a bibulous support therebytransporting reagents and samples to sites on the support and/or washingthe support. Such devices have been described by Deutsch, U.S. Pat. No.4,094,647; Zuk et al, U.S. Pat. No. 4,435,504; Weng et al, U.S. Pat. No.4,740,468; Friesen et al, German Offenlengungschrift 3,445,816 etc.

In many of these methods, reagents can be added at more than oneposition on the bibulous support. In such situations, it is oftendesirable to cause the added reagent to migrate in only one directionalong the bibulous support, to delay flow in a given direction, or tocreate a direction of flow that was impeded prior to the addition of thereagent. For example, it is often desirable for a solution moving alonga bibulous support to automatically stop moving after a certain volumehas been taken up so as to avoid the necessity to monitor the flow ofsolution. Subsequent to taking up a certain volume, a second solution isallowed to flow along the support. In the past, the first flow could bestopped by limiting the length of the first bibulous support and thesecond flow initiated by contacting one end of the first bibuloussupport with a solution and the other end with a second bibulous supportwhich is dry.

Likewise, in liquid containing assay devices, a sample is added to oneof the ports and liquid within the device washes the sample away from adetection zone. See, for instance, Khanna et al, U.S. patent applicationSer. No. 35,562 filed Apr. 7, 1987 entitled "Immunoassay Devices". Tomaximize the washing efficiency, it is desirable that upon contactingthe support with the sample, the sample flows in only the direction awayfrom the source of the wash liquid.

In both of the above cases, it is desirable to create a capillary flowrelationship between pieces of bibulous material which heretofore havebeen in a non-capillary flow relationship. Moreover, it would beparticularly desirable to create such a capillary flow relationshipautomatically without mechanical means external to the device whilerequiring minimal operator involvement to operate.

U.S. Pat. No. 3,482,943 discloses expandable sponges useful intransporting solution to a set position on a gel suitable for conductingimmunodiffusion tests. In this reference, the solution transported bythe sponge is allowed to diffuse into the gel which, in order to conductthe immunodiffusion, is by necessity a wet gel. Accordingly, nocapillary flow relationship between the expandable sponge and the gel isestablished by this device.

U.S. Pat. No. 4,246,339 discloses a device having an upper portion and alower portion. The upper portion has a plurality of wells wherein thebottom of each well is fitted with a membrane layer capable oftransporting liquid. The bottom portion of the device contains absorbentmaterial. Between the top and bottom portions is a compressible spacer.This device allows a liquid sample to be added to the wells which mayoptionally be impregnated with an antibody. After a set incubationperiod, pressure is placed on the top portion which because of thecompressible spacer, allows the bottom of each of the wells to contactthe absorbent material whereupon the liquid in the wells is transferredto the absorbent material. After liquid transfer, the pressure isremoved and contact between the bottom of the wells and the absorbentmaterial is broken. Thereupon, additional liquid may be added to thewells. In this device, care must be taken to insure that the bottom ofall of the wells come into contact with the absorbent material for asufficient period of time to remove the liquid. In particular, if one ormore of the wells does not contact the absorbent material, then theliquid in that well will not be removed. Moreover, if one or more of thewells does not contact the absorbent material for a sufficient period oftime, then not all of the liquid in that well will be removed. In anycase, a high level of operator care is required to ensure the properoperation of this device.

U.S. Ser. No. 35,562 filed Apr. 7, 1987 entitled "Immunoassay Devices"discloses a device for conducting an assay method. The device comprisesa housing having in one portion thereof a breakable capsule and inanother portion a piece of bibulous material attached to an absorbentpad.

European Patent Application Publication No. 0 146 691 discloses an airbleed passage in a liquid sampling needle which is formed between ahousing and a sleeve and into which is added a solid compacted materialwhich swells on contact with liquid. As the liquid sample is drawn intothe needle, air can escape via the air bleed until the air bleed iscontacted with liquid whereupon it expands and forms a liquidimpermeable membrane.

U.S. Pat. No. 4,700,741 discloses a urine collecting device whichcontains an expandable sponge in a compartment which permits thecollection of a predetermined quantity of urine by limiting expansion ofthe sponge within the compartment.

Canadian Patent No. 1,185,882 discloses porous, hydrophilic,non-gel-forming swellable polymers or self-drawing fluid reservoirs witha very high and uniform absorption and release of fluid in achromatographic quick-test device.

U.S. Pat. No. 4,826,759 describes apparatuses and methods, which can beused in the field (i.e., outside the laboratory environment) todetermine qualitatively and at least semiquantitatively the presence orabsence of minute quantities of ligand. The apparatus can be in the formof a strip comprising a support means provided with a grooveintermediate its ends forming a crease line upon which the strip can befolded upon itself with bibulous elements and spaced from the creaseline and arranged so that when the strip is folded upon itself thebibulous elements become aligned with each other and come into liquidcontact.

U.S. Pat. No. 4,803,170 discusses an immunoassay device including one ormore reaction chambers, each adapted to receive and retain a volume oftest fluid communication with non-overlapping first, second, and thirdreagent-bearing surfaces. To the first surface is reversibly bound ananalyte conjugate: Analyte component conjugated to one or morecomponents, termed ligand/marker, that serve ligand and marker functionsas described herein. Analyte binding partner is immobilized on thesecond surface, and ligand/marker binding partner is immobilized on thethird surface. The reaction chamber is preferably configured to receiveand direct the test fluid sequentially past the first, second, and thirdreagent surfaces. In use, analyte conjugate solubilized from the firstsurface competes with any analyte in the test fluid for analyte bindingpartner sites on the second surface. Excess analyte conjugate becomessequestered on the third surface, where the marker activity is read toindicate analyte presence and concentration in the test fluid. A testkit includes the immunoassay device in combination with comparative testresults.

U.S. Pat. No. 3,888,629 describes a reaction cell for the performance ofradioimmunoassay determinations and like saturation analysis reactionsthat has supported within it a matrix pad of absorbent material capableof retaining the necessary reagents for the reaction and serving as asite in which the reaction totally occurs. A separable lower chamber isfitted to the lower end of the cell and contains absorbent material toabut the matrix pad and promote filtration through the pad after thereaction has taken place. An upper reservoir chamber fits to the upperend of the cell to contain liquid for passing through the matrix pad.The matrix pad will commonly contain prior to the reaction apredetermined amount of an antigen or antibody in freeze-dried conditionand possibly radioactivity labelled.

Accordingly, there is a need for a device for creating a capillary flowrelationship between pieces of bibulous material which prior toactuation, are in a non-capillary flow relationship to each other.Preferably, such a device should require minimal operator involvement tooperate.

SUMMARY OF THE INVENTION

The present invention is directed to assay apparatus, devices andmethods that permit longitudinal capillary flow of liquid through twopieces of bibulous material, which prior to actuation are in anon-capillary flow relationship to each other. In particular, in one ofits device aspects, the present invention is directed to a device foruse in an assay that permits capillary flow of a liquid through twopieces of bibulous material which prior to actuation are in anon-capillary flow relationship to each other which comprises (a) twopieces of bibulous material in a non-capillary flow relationship to eachother and (b) a distortable member, which when distorted actuates thedevice by creating a longitudinal capillary flow relationship betweenthe two pieces of bibulous material. The distortable member may haveaffixed thereto a third piece of bibulous material in a non-capillaryflow relationship with the two pieces of bibulous material in (a) aboveor the distortable member may comprise a portion of one of the twopieces of bibulous material.

In one of its method aspects, the present invention is directed toward amethod for carrying out an assay that comprises using a longitudinalcapillary flow relationship between two pieces of bibulous material,which prior to creating the relationship are in a non-capillary flowrelationship to each other. The method comprises the step of contactingtwo pieces of bibulous material, each in a non-capillary flowrelationship to the other, with a distortable member so as to achieve alongitudinal capillary flow relationship between the pieces. Thedistortable member may comprise a portion of one of the two pieces ofbibulous material. Alternatively, a third piece of bibulous material canbe affixed to the distortable member and contacting of the pieces can beobtained by distorting the distortable member.

In another embodiment the distortable member may include a portion ofone of the two pieces of bibulous material. The two pieces of bibulousmaterial may be affixed to a support, where a portion of one of the twopieces is not supported by the support and is therefore distortable. Thesupport can be adapted for insertion into a housing. One or moreflexible members, may be attached to the housing. In this embodiment thesupport is inserted into the housing and one of the flexible membersforces the distortable portion, usually an adjacent end of one of thetwo pieces of bibulous material, into contact with an adjacent end ofthe other.

In another of its method aspects, the present invention is directedtoward a method for carrying out an assay by creating a longitudinalcapillary flow relationship between two pieces of bibulous materialwhich prior to actuation are in a non-capillary flow relationship toeach other which comprises (a) providing a device which comprises (i)two pieces of bibulous material in a non-capillary flow relationship toeach other, and (ii) a distortable member, and (b) distorting thedistortable member which thereby results in contact of the pieces ofbibulous material so as to achieve a longitudinal capillary flowrelationship between the pieces of bibulous material.

The invention further includes kits and apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of one embodiment of this invention inits unactuated state.

FIG. 2A illustrates the device depicted in FIG. 1 in an actuated state.

FIG. 2B illustrates the device depicted in FIG. 1 in an actuated stated.

FIG. 3 is a perspective view of another embodiment of this invention.

FIG. 4A is a cross-sectional view of the device of FIG. 3 taken alonglines 4--4 of FIG. 3 and illustrates this device in an unactuated state.

FIG. 4B illustrates the device depicted in FIG. 4A in an actuated state.

FIG. 5A is a side view of another embodiment of a device in accordancewith the present invention.

FIG. 5B is a front view of the device shown in FIG. 5A.

FIG. 6A is a front view of an enclosure for the device of FIGS. 5A and5B.

FIG. 6B is a side view of the enclosure of FIG. 6A.

FIG. 7 is an exploded perspective view of a housing and a container forthe device of FIGS. 6A and 6B.

FIG. 8 is a top plan view of the housing shown in FIG. 7.

FIG. 9 is a side elevational view depicting the insertion of the deviceof FIGS. 6A and 6B into the housing and container of FIGS. 7 and 8.

FIGS. 9A-9C are enlarged cross-sectional views showing the progressionof the insertion of the device of FIGS. 6A and 6B into the housing andcontainer of FIGS. 7 and 8.

DETAILED DESCRIPTION OF THE INVENTION

Assay devices, apparatus and methods are provided which permit theformation of a capillary flow relationship between two pieces ofbibulous material. In particular, the devices of the present inventionare useful for assays in which a longitudinal capillary flowrelationship is created between two pieces of bibulous material whichtheretofore were in a non-capillary flow relationship to each other.

The devices of the present invention are adaptable to a wide variety ofanalytical uses which require a capillary flow relationship betweenpieces of bibulous materials which prior to actuation, were in anon-capillary flow relationship. The devices of the present inventionare particularly suited for use in conjunction with chromatographicmethods, particularly those providing assays for biologicallysignificant analytes. The devices are particularly suited for clinicalassay methods including immunoassays such as radioimmunoassays, enzymeimmunoassays, fluorescent immunoassays, etc.

Before proceeding further with the description of the specificembodiments of the present invention, a number of terms will be defined.

Bibulous material--a porous material having pores of at least 0.1 μm,preferably at least 1.0 μm, which is susceptible to traversal by aliquid medium, for example, an aqueous medium, in response to capillaryforce. Such materials are generally hydrophilic or hydrophobic dependingon whether the liquid medium is polar or non-polar, respectively, or arecapable of being rendered hydrophilic or hydrophobic and includeinorganic powders such as silica, magnesium sulfate, and alumina;natural polymeric materials, particularly cellulosic materials andmaterials derived from cellulose, such as fiber containing papers, e.g.,filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as polystyrene, polyethylene,nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide,cross-linked dextran, agarose, polyacrylate, etc.; either used bythemselves or in conjunction with other materials; ceramic materials;and the like. The bibulous material can be attached to a support. On theother hand, the bibulous material may provide its own support. Thebibulous material may be polyfunctional or be capable of beingpolyfunctionalized. The bibulous material employed in the presentinvention is not susceptible to expansion when exposed to liquid. Thatis to say when wetted, the bibulous material will not expand by morethan about 10%; although preferably by no more than about 5%.

The pieces of bibulous material can be a single structure such as asheet cut into strips or it can be several strips or particulatematerial bound to a support or solid surface such as found, for example,in thin-layer chromatography and may have an absorbent pad either as anintegral part or in liquid contact. The piece of bibulous material canbe comprised of several segments, one or more being an absorbent pad,bound to a support. The piece of bibulous material can also be a sheethaving lanes thereon or capable of spotting to induce lane formation,wherein a separate assay can be conducted in each lane. The absorbentpad may be any hydrophilic bibulous material such as paper, sponge,felt, porous polymers and the like. The piece of bibulous material canhave a rectangular, circular, oval, triagonal or other shape providedthat there is at least one longitudinal direction of traversal of aliquid test solution by capillary migration that is at least 10 timesthe thickness of the piece and is preferably at least 20 or more timesthe thickness of the piece. Such a situation gives rise to alongitudinal capillary flow relationship as the term is used herein. Inthe following discussion, strips of bibulous material will be describedby way of illustration and not limitation.

The support for the bibulous material, where a support is desired ornecessary, will normally be insoluble in the liquid medium, non-porous,and rigid and usually will be of the same length and width as thebibulous strip but may be larger or smaller. A wide variety of organicand inorganic materials, both natural and synthetic, and combinationsthereof, may be employed provided only that the support does notinterfere with the capillary action of the strip, or, in the case wherethe bibulous material is used in an assay, non-specifically bind assaycomponents, or interfere with the signal produced by the assay.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutane), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), glass, ceramics, metals,and the like.

The pieces of bibulous material used in the present invention need notbe comprised of the same bibulous substance. For example, in anembodiment utilizing three pieces of bibulous material, two of thepieces of bibulous material can be comprised of silica whereas the thirdpiece of bibulous material can be comprised of alumina. However, in apreferred embodiment, all of the pieces of bibulous material arecomprised of the same bibulous substance.

Non-capillary flow relationship--two pieces of bibulous material are ina non-capillary flow relationship when liquid is unable to move bycapillarity, i.e., capillary migration, from the first piece to thesecond piece of bibulous material. One manner of creating anon-capillary flow relationship between two pieces of bibulous materialis to prevent any contact between the pieces. On the other hand, the twopieces may be in contact with each other but capillary flow between thepieces may be prevent by a physical or chemical barrier. In this case,these two pieces are in a non-capillary flow relationship to each other.

Longitudinal capillary flow relationship--two pieces of bibulousmaterial are in a longitudinal capillary flow relationship when liquidis able to move by capillarity, i.e., capillary migration, from onepiece to the other piece of bibulous material and thereby provide forcapillary flow along a long dimension of at least one of the pieceswherein the long dimension is at least 10 times the length of theshortest dimension of the piece measured perpendicular to the directionof flow. As shown by the present invention, one manner of creating alongitudinal capillary flow relationship between two pieces of bibulousmaterial is to utilize a third piece of bibulous material which contactsboth the first and second piece of bibulous material so as to complete afluidic circuit among all three pieces of bibulous material. Anothermanner of creating a longitudinal capillary flow relationship betweentwo pieces of bibulous material is to employ a distortable member toforce adjacent ends of the pieces into contact with each other.

Distortable member--a member which under applied pressure is capable ofbeing bent, constricted or compressed or otherwise moved to cause twopieces of bibulous material to come into intimate contact and therebyinitiate a longitudinal capillary flow relationship between the twopieces of bibulous material. At least a portion of the distortablemember lies substantially opposite the adjacent ends of the two piecesof bibulous material, i.e., the ends that will be involved in formingthe capillary flow relationship between the two pieces of bibulousmaterial. The two pieces of bibulous material are generally affixed tothe same surface, but need not be. The distortable member can have athird piece of bibulous material affixed thereto. Where the device doesnot include a third piece of bibulous material, the distortable membercan comprise a portion of one of the two pieces of bibulous material.

In one embodiment, the distortable member is part of a device having twoarms, wherein the arms preferably lie substantially parallel to eachother and are connected to each other through a central shoulder. Inthis embodiment, at least one of the arms is capable of beingconstricted or compressed or bent toward the other arm when a force isapplied to that arm with a directional component running substantiallyperpendicular to and toward the other arm. Also, in this embodiment, thedistortable member is that arm which has a piece of bibulous materialwhich contacts the two other pieces of bibulous material regardless ofwhich arm actually moves. Thus, the distortable member may have itsposition fixed by attachment to a wall or support and the other arm maymove in relationship to the distortable member or the other arm may beattached to a wall and the distortable member may move. However, ineither case, upon distortion, the piece of bibulous material on thedistortable member will contact the two other pieces of bibulousmaterial on the other arm thereby initiating a capillary flowrelationship among all three pieces of bibulous material.

In this embodiment, the distortable member is attached to a shoulderwhich is comprised of a flexible material such as a semi-rigid plasticor a pliable metal which allows at least one of the arms of thedistortable member to be susceptible to compression (in the direction ofthe other arm) by applied pressure. Generally, the flexible materialshould be sufficiently pliable so as to result in compression of atleast one of the arms by the pressure resulting from squeezing, etc. Ingeneral, two pieces of bibulous material are positioned on one arm (in anon-capillary flow relationship to each other) and the third piece ofbibulous material is positioned on the distortable member (other arm) insuch a manner that upon distortion, the third piece of bibulous materialcontacts both the two other pieces of bibulous material so as to resultin the formation of a capillary flow relationship among the threepieces.

In another embodiment, the distortable member having a third piece ofbibulous material affixed thereto is part of a device wherein the twopieces of bibulous material are attached to a surface of a supporthaving a fixed position. In this embodiment, the distortable member iscapable of constricting or compressing or bending in the direction ofthis support. Such constriction is generally the result of movement byapplying a force to the distortable member around a shoulder comprisedof flexible material or a hinge.

In another embodiment the distortable member is a portion of one of thetwo pieces of bibulous material. The two pieces of bibulous material arefixedly attached to a support where a portion of one of the piecesextends beyond the support. The support is adapted for insertion into ahousing having one or more flexible members. In a method in accordancewith the invention the support is inserted into the housing. Thedistortable portion of one of the pieces of bibulous material bends,when contacted by a flexible member, and is forced to contact anadjacent end of the other piece of bibulous material. As a result alongitudinal capillary flow relationship is initiated between the twopieces of bibulous material, thus achieving actuation of the device.

Analyte--the compound or composition to be measured that is capable ofbinding specifically to an antibody, usually an antigen or drug.

The precise nature of the antigenic and drug analytes together withnumerous examples thereof are disclosed in U.S. Pat. No. 4,299,916 toLitman, et al., particularly columns 16 to 23, and in U.S. Pat. No.4,275,149, columns 17 and 18, the disclosures of which are incorporatedherein by reference.

The analytes are characterized by having single binding sites(monovalent) or multiple binding sites (polyvalent). The polyvalentanalytes will normally be poly(amino acids), i.e., polypeptides andproteins, polysaccharides, nucleic acids, and combinations thereof. Suchcombinations or assemblages include bacteria, viruses, chromosomes,genes, mitochondria, nuclei, cell membranes, and the like.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes of interest include drugs, metabolites, pesticides,pollutants, and the like.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, DNA, RNA,thyroxine binding globulin, thyroxine binding prealbumin, transcortin,etc.

Member of a specific binding pair ("sbp member")--one of two differentmolecules having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin hormones-hormone receptors, nucleic acid duplexes,IgG-protein A, DNA-DNA, DNA-RNA, and the like are not immunologicalpairs but are included in the definition.

Ligand--any organic compound for which a receptor naturally exists orcan be prepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component Clq. and the like.

Labeled sbp member--a label, generally capable of electrochemicaldetection or absorption or emission of electromagnetic radiation, acatalyst, frequently an enzyme, bound to a first sbp member. The labeledsbp member is a member of the signal producing system and the first sbpmember is chosen to bind to the second sbp member in accordance with aparticular protocol in an assay.

Antibody--an immunoglobin, or derivative or fragment thereof, having anarea on the surface or in a cavity which specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as, for example, immunization of a host and collection of seraor hybrid cell line technology.

Antibody for the analyte--an antibody specific for an analyte.

First sbp member--a modified analyte or analyte analog or surrogatewhich can compete with the analogous analyte in binding to a second sbpmember, usually a receptor or antibody, the modification providing meansto join the analyte analog to a label to provide a labeled sbp member.The analyte analog will usually differ from the analyte by more thanreplacement of a hydrogen with a bond which links the analyte analog toa hub or label, but need not. The term analyte surrogate refers to acompound having the capability of binding the antibody for the analyte.Thus, the analyte surrogate may bind to the antibody for the analyte ina manner similar to the analyte. On the other hand, the surrogate couldbe, for example, an antibody directed against the idiotype of anantibody to the analyte.

The first sbp member can also be a conjugate of an analyte, a label,e.g., enzyme, and a third sbp member.

Second sbp member--an sbp member capable of binding to the analyte andthe first sbp member. The second sbp member can bind to a determinantsite on the analyte and to a determinant site on the first sbp member. Apreferred second sbp member is an antibody.

Binding agent--a material non-diffusively bound to the situs capable ofbinding the first sbp member. The material may bind non-specifically orspecifically. When the binding is non-specific, the binding agent willusually be hydrophobic or polyionic. When the binding agent provides forspecific binding, it will usually be an sbp member complementary to thelabeled sbp member, preferably a receptor for the first sbp member. As apractical matter, the binding agent will usually be the second sbpmember, but it may be either member of a large variety of specificbinding pairs, provided only that the complementary member is bound tothe labeled sbp member. When the first sbp member is a conjugatecomprising an analyte, a label, and a third sbp member, the bindingagent can be a corresponding receptor for the third sbp member.

Label--A label may be any molecule bound to the first member that isrequired to produce a signal. In the subject invention, the label may beinert and serve solely as a binding site for a member of the signalproducing means or it may spontaneously produce a detectable signal ormay produce a detectable signal in conjunction with a signal producingmeans. The label may be isotopic or nonisotopic, preferably nonisotopic.However, an isotopic label can be preferred for achieving highsensitivity when using radio-autographic detections with photographicfilm.

Signal producing means--means capable of interacting with the label toproduce a detectible signal. Such means include, for example,electromagnetic radiation, heat, chemical reagents, and the like. Wherechemical reagents are employed, some of the chemical reagents can beincluded as part of a developer solution. The chemical reagents caninclude substrates, coenzymes, enhancers, second enzymes, activators,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Some of the chemical reagents such as coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the likecan be bound to the strip.

Signal producing system--The signal producing system may have one ormore components, at least one component being the labeled sbp member.The signal producing system includes all of the reagents required toproduce a measurable signal including signal producing means capable ofinteracting with the label to produce a signal.

The signal producing system provides a signal detectable by externalmeans, normally by measurement of electromagnetic radiation, desirablyby visual examination. For the most part, the signal producing systemincludes a chromophoric substrate and enzyme, where chromophoricsubstrates are enzymatically converted to dyes which absorb light in theultraviolet or visible region, phosphors or fluorescers.

Ancillary Materials--Various ancillary materials will frequently beemployed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers. Frequently, in addition to these additives, additionalproteins may be included, such as albumins, or surfactants,particularly, non-ionic surfactants, binding enhancers, e.g.polyalkylene glycols, or the like.

Referring now to the drawings, FIG. 1 is a cross-sectional view of oneembodiment of this invention. In FIG. 1, device 10 contains two arms 12and 14 attached through shoulder 16. Arm 12 contains a first and secondpiece of bibulous material 18 and 20, respectively; whereas arm 14contains a piece of bibulous material 22. Arm 14, which contains onlyone piece of bibulous material, is also referred to as the distortablemember. Because none of the pieces of bibulous material are in acapillary flow relationship with any other piece of bibulous material,device 10 is in its unactuated state. The bottom portion of piece ofbibulous material 18 of device 10 is designed to be inserted into acontainer 24 having a liquid solution 26 which can traverse bycapillarity at least that portion of piece of bibulous material betweenliquid solution 26 and the portion of bibulous material 18 capable ofcontact with bibulous material 22.

FIG. 2A illustrates one method of actuating the device of FIG. 1. Inparticular, the bottom portion of piece of bibulous material 18 ofdevice 10 is generally inserted into a second container 28 with a liquidsolution 30 and is actuated by applying inward pressure on the outersurfaces of arms 12 and 14. Sufficient force is applied to ensure thatpiece of bibulous material 22 is brought into contact with both the topportion of piece of bibulous material 18 and the bottom portion of pieceof bibulous material 20. In general, sufficient force can be appliedeither mechanically or manually, preferably mechanically, by squeezingtogether arms 12 and 14. Mechanical means of applying sufficient forceinclude squeezing arms 12 and 14 by insertion into a slot in a devicedesigned to provide a uniform pressure at the bibulous material contactsurfaces sufficient to cause liquid flow without compressing thebibulous material to such an extent that flow is restricted. Shoulder 16of device 10 is comprised of a flexible material which allows the armsto be susceptible to compression by applied pressure. The flexiblematerial should be sufficiently pliable so as to result in compressionwhen pressure is applied. Suitable flexible materials include semi-rigidplastics and pliable metal.

FIG. 2B illustrates another method of actuating the device of FIG. 1. Inparticular, device 10 is actuated by applying inward pressure on theouter surfaces of arms 12 and 14. Sufficient pressure is applied toensure that piece of bibulous material 22 is brought into contact withboth the top portion of piece of bibulous material 18 and the bottomportion of piece of bibulous material 20. In FIG. 2B, device 10 isactuated by inserting this device into container 32 having wall 34,protrusion 36 and liquid solution 38. By virtue of limited space incontainer 32, arms 12 and 14 are constricted or compressed toward eachother which results in piece of bibulous material 22 contacting piecesof bibulous material 18 and 20. When such contact is made, all threepieces of bibulous material are in a capillary flow relationship to eachother.

Arms 12 and 14, container 32 as well as protrusion 36 are generallycomprised of any non-corrodable materials which do not readily degradeor disintegrate upon exposure to the solutions employed in the assay ortest. Additionally, such materials should not interfere with the assayor test being conducted. In general, metals, metal alloys, glass andrigid and semi-rigid plastics can be used. Preferably, glass or a rigidplastic is employed. As used herein, the term "non-corrodable" meansthat the material is not subject to undo decomposition or disintegrationwhen routinely used in the devices of the present invention.

Device 10 is generally operated in the following manner; the bottomportion of bibulous material 18 of device 10 is inserted into a firstsolution 26. This solution is allowed to traverse all or part ofbibulous material 18 by capillarity. Because piece of bibulous material18 is not in a capillary flow relationship with either piece of bibulousmaterial 20 or 22, once solution 26 reaches the top end of piece ofbibulous material 18, capillary flow ceases. The wetted bibulousmaterial may then be incubated in contact with solution 26 and/or device10 may be withdrawn from solution 26 and the bottom portion of piece ofbibulous material 18 is inserted into solution 30 or 38 which ispreferably a solution different from solution 26. In the situation setforth in FIG. 2A, device 10 is then actuated which results in piece ofbibulous material 22 contacting both piece of bibulous material 18 andpiece of bibulous material 20 thereby creating a longitudinal capillaryflow relationship among all three pieces of bibulous material. Becausepieces of bibulous material 22 and 20 are dry, capillary flow will bereintiated until either the solution is exhausted or all of the piecesof bibulous material are completely wetted. Bibulous materials 18 and/or20 can have appropriate reagents bound at predetermined sites thereon.See, for instance, Zuk et al, U.S. Pat. No. 4,435,504; Weng et al, U.S.Pat. No. 4,740,468; and Tom et al, U.S. Pat. No. 4,366,241, thedisclosure of each of which is incorporated herein in their entirety byreference.

In the situation set forth in FIG. 2B, device 10 is actuated byinsertion into container 32 which results in piece of bibulous material22 contacting both piece of bibulous material 18 and piece of bibulousmaterial 20 thereby creating a longitudinal capillary flow relationshipamong all three pieces of bibulous material. Because pieces of bibulousmaterial 22 and 20 are dry, capillary flow will be reintiated untileither the solution is exhausted or all of the pieces of bibulousmaterial are completely wetted.

In another embodiment not shown in either FIG. 2A or 2B, an absorbentpad may be in liquid receiving contact, i.e., in capillary flowrelationship, with the upper terminus of piece of bibulous material 20.This embodiment permits greater quantities of liquid to be moved bycapillarity along pieces of bibulous material 18, 22 and 20.

FIG. 3 is a perspective view of another embodiment of a device of thepresent invention. In FIG. 3, device 40 is encased by housing 42 whichin turn has portal 44 which allows a liquid sample to be placed on pieceof bibulous material 54 (shown in FIGS. 4A and 4B). Device 40 has a topwall 46 which has a distortable member 48 and hinge 50. Hinge 50 allowsdistortable member 48 to move under applied pressure downward inrelationship to top wall 46.

FIG. 4A illustrates in cross-section the interior of device 40 in itsunactuated state. Device 40 contains a bottom surface 52 on the top ofwhich is attached a first piece of bibulous material 54 and a secondpiece of bibulous material 56. Pieces of bibulous material 54 and 56 donot contact each other and accordingly, are in a non-capillary flowrelationship to each other. Second piece of bibulous material 56 is in acapillary flow relationship with an absorbent pad 58. Downwardprotrusion 60 of top wall 46 form conical portal 44 which is preferablyin contact with piece of bibulous material 54 and facilitates theaddition of liquid sample (not shown) to piece of bibulous material 54.While portal 44 is conical in nature in FIG. 4A, it need not be.Distortable member 48 has a downward protrusion 62 which terminates inpiece of bibulous material 64 which is positioned to overlap both firstand second pieces of bibulous material 54 and 56. Distortable member 68has frangible connection 49 with top wall 46. In its unactuated state,piece of bibulous material 64 does not contact either first or secondpiece of bibulous material 54 and 56 respectively and therefore allthree pieces of bibulous material are in a non-capillary flowrelationship to each other.

FIG. 4B illustrates in perspective the interior of device 40 in itsactuated state. As shown in this figure, device 40 is actuated by thedownward movement of distortable member 48 wherein frangible connection49 is broken. The downward movement of 48 results in piece of bibulousmaterial 64 contacting both first and second piece of bibulous material54 and 56 respectively thereby creating a capillary flow relationshipamong all three pieces of bibulous material. Distortable member 48 ismoved downward through hinge 50 by use of applied downward pressure suchas that resulting from a thumb or finger pushing downward on distortablemember 48. When wall 47 of distortable member 48 moves past point 49,member 48 is held in position because top wall 46 extends slightly overmember 48 at point 49.

In operation, device 40 is utilized by first placing a liquid sample onbibulous material 54 through portal 44. Where protusions 60 formingportal 44 are in contact with piece of bibulous material 54, portal 44directs the liquid sample to the appropriate position on piece ofbibulous material 54. Alternatively, if portal 44 is not in contact withpiece 54 but merely is an opening in top wall 46, then the liquid samplecan be added to piece of bibulous material 54 by inserting a syringe orpipette through portal 44. Preferably, portal 44 is positioned at orvery near the end of bibulous material 54 furthest away from piece ofbibulous material 56. The amount of liquid sample should be kept to nogreater than the amount that can be absorbed by piece of bibulousmaterial 54. Bibulous materials 54 and/or 56 can have appropriatereagents bound at predetermined sites thereon. See, for instance, Zuk etal, U.S. Pat. No. 4,435,504; Weng et al, U.S. Pat. No. 4,740,468; andTom et al, U.S. Pat. No. 4,366,241, the disclosure of each of which isincorporated herein in their entirety by reference. After application ofliquid sample to piece of bibulous material 54, any additional reagentscan be added via portal 44. In this regard, after piece of bibulousmaterial 54 has been completely wetted, device 10 can be actuated byapplying pressure on distortable member 48 which results in piece ofbibulous material 64 contacting both piece of bibulous material 54 andpiece of bibulous material 56. Because pieces of bibulous material 56and 64 are dry and further because piece of bibulous material 56 is in alongitudinal capillary flow relationship with absorbent pad 58, acapillary flow relationship is initiated among all pieces of bibulousmaterial which will continue until either the solution is exhausted orall of the pieces of bibulous material (including the absorbent pad) arecompletely wetted. Accordingly, after actuation of device 40, additionalliquid reagents, i.e., wash solutions, solutions containing reagentsrequired in the assay or test, etc., can be added to piece of bibulousmaterial 54 via portal 44 and transported along by capillary action.

Alternatively, additional liquid reagents can be added to piece ofbibulous material 54 by use of a breakable capsule such at thatdisclosed in U.S. Ser. No. 35,562 filed Apr. 7, 1987 entitled"Immunoassay Device" and which is incorporated herein in its entirety byreference. Yet another method of adding additional liquid to piece ofbibulous material 54 can be accomplished by use of an expandable pieceof bibulous material either by itself or in conjunction with a breakablecapsule as disclosed in our patent application entitled "Device andMethod for Completing a Fluidic Circuit Which Employs a LiquidExpandable Piece of Bibulous Material" which is to be filed concurrentlywith the instant application and which is incorporated herein in itsentirety by reference. In such a case, device 40 would be modified so asto incorporate a liquid expandable piece of bibulous material andoptionally, a breakable capsule.

In a preferred embodiment, top wall 46 of device 40 contains a means toview all or a portion of bibulous material 54. One means for viewingbibulous material 54 is by having an appropriate window in top wall 46.Such a window can take the form of a glass or clear plastic encompassingall of top wall 46 or only that portion of top wall 46 which exposes therelevant portion of bibulous material 54. In this regard, many assayswhich utilize bibulous material have a small detection zone relative tothe size of bibulous material employed which upon completion of theassay, indicates the presence or absence of the material being testedfor. Accordingly, in those circumstances, it is not necessary to viewthe entire length of bibulous material 54 but only necessary to view thedetection zone. On the other hand, some assays require that the entirebibulous material be viewed in order to quantitatively determine thepresence of analyte. The window can also take the form of an opening intop wall 46 which allows viewing of the relevant portion of bibulousmaterial 54.

Referring now to FIGS. 5A and 5B rigid support 80 has fixedly attachedthereto first strip of bibulous material 82 having thereon a series ofzones 84 in each of which are bound sbp members. Support 80 turns backon itself at portion 86 to yield portion 88 that has second strip ofbibulous material 90 fixedly attached thereto. Second strip 90 facestowards first strip 82 and has distortable portion 92, which extendsbelow portion 88 of support 80 and serves as a distortable member.

Support 80 is encased in enclosure 94 (FIGS. 6A and 6B), open at thebottom 96 to permit entry of liquid. Enclosure 94 also has openings 98in recessed portion 100. Openings 98 permit viewing of zones 84 todetermine the results of an assay utilizing this particular embodimentof the invention. Enclosure 94 also has opening 102, which allows accessto distortable portion 92 of second strip of bibulous material 90 sothat distortable portion 92 can be bent to contact portion 104 of firststrip of bibulous material 82. Enclosure 94 also has openings 93 and 95for viewing first strip of bibulous material 82.

FIG. 7 depicts housing 106 having flexible member 108 fixedly attachedto inner wall 110 of housing 106. Flexible member 112 (FIG. 8) isfixedly attached to inner wall 114 of housing 106 opposite flexiblemember 108. The function of flexible members 108 and 112 will beexplained hereinbelow. Housing 106 is adapted to attach to the top ofcontainer 116.

Referring now to FIGS. 9 and 9A-9C, enclosure 94 depicted in FIG. 6A isdesigned to be inserted into housing 106, which has been fitted intocontainer 116. As enclosure 94 is inserted into housing 106, the portionof enclosure 94 at opening 96 contacts flexible member 108 and flexiblemember 112. The rigidity of enclosure 94 causes members 108 and 112 tobe bent downward (FIG. 9B) thus allowing insertion of enclosure 94. Asenclosure 94 moves further into container 116, opening 102 in enclosure94 is engaged by flexible member 108, which flexes inwardly to contactdistortable portion 92 of second strip of bibulous material 90 (FIG.5A). Flexible member 108 forces distortable portion 92 to contactportion 104 of first strip of bibulous material 82. As a result acapillary flow relationship in initiated between first and second piecesof bibulous material, 82 and 90, respectively. A liquid medium (notshown) in the bottom of container 116 contacts first strip of bibulousmaterial 82, along which it traverses by capillarity into and alongsecond strip of bibulous material 90.

In view of the above, it is apparent that devices of the presentinvention allow the creation of a longitudinal capillary flowrelationship among two or three pieces of bibulous material whichtheretofore were in a non-capillary flow relationship. Such devices havethe particular advantage of allowing the device operator to choose thetime to actuate the device and thus create the capillary flowrelationship.

The housing used in some of the devices of the present invention as wellas the bases and protrusions can be prepared from non-corrodablematerials which do not readily degrade or disintegrate upon exposure tothe solutions employed in the assay. Additionally, such materials shouldnot interfere with the assay being conducted. In general, metals, metalalloys, glass and rigid and semi-rigid plastic can be used. Preferably,a rigid or a semi-rigid plastic is employed.

The dimensions of the devices of the present invention can varydepending on the particular use, that is, whether the devices are usedin immunoassays, etc. The length as measured in the direction ofcapillary flow of at least one of the pieces of bibulous materialemployed in a device of the present invention will generally be at leastabout 10 times the shortest dimension measured perpendicular to thedirection of capillary flow and may be 20 or more times the shortestdimension. Other dimensions may be varied considerably depending uponfactors such as whether an absorbent pad is employed, the amount ofliquid required to be taken up by the assay, whether a wash solution isrequired or desired for the assay, etc. Likewise, the extent which theconstrictable member must compress depends on factors such as the spacebetween the piece of bibulous material affixed to the constrictablemember and the two other pieces of bibulous material. Those skilled inthe art will be able to construct devices of the present inventionhaving appropriate dimensions in view of the disclosure herein.

The devices of the present invention can be employed to determine, forexample, the result of a chemical test particularly by employing achromatographic step. The present device can find application in amethod for determining qualitatively and/or quantitatively the presenceor amount, respectively, of an analyte in a sample suspected ofcontaining the analyte. Examples of such methods are described in U.S.Pat. Nos. 4,366,241; 4,740,488; 4,168,146 and 4,435,504, the disclosuresof which are incorporated herein by reference and the terms used belowhave the meanings set out therein, which meanings have been summarizedabove.

The above methods may be carried out on, among others, a bibulous stripas a stationary solid phase and involving a moving liquid phase. Thestationary solid phase can be contacted with a plurality of reagents ina number of different solutions.

Referring to FIG. 2B, for an assay for analyte a member of specificbinding pair (sbp member) can be non-diffusively bound to a bibulousstrip to form an "immunosorbing zone" which may be all or a portion ofpiece of bibulous material 18 or 20. The analyte from the sampletraverses piece 18 being carried along with a solvent whose frontcrosses the zone. The analyte, which may be the homologous or reciprocalsbp member to the sbp member bound to the support, becomes bound to thesupport through the intermediacy of sbp member complex formation. Thesignal producing system provides the manner by which part or all of thearea in the immunosorbing zone to which the analyte is bound may bedistinguished from the area in which it is absent.

The incremental movement of the sample through the immunosorbing zoneresults from the transport of a solution of the sample in an appropriatesolvent through the immunosorbing zone due to capillarity.

The solvent is normally an aqueous medium, which may be up to about 40weight percent of other polar solvents, particularly oxygenated solventsof from 1 to 6, more usually of from 1 to 4 carbon atoms, includingalcohols, ethers and the like. Usually, the co-solvents are present inless than about 20 weight percent.

The pH for the medium is usually in the range of 4-11, more usually5-10, and preferably in the range of about 6.5-9.5. The pH is chosen tomaintain a significant level of binding affinity of the sbp members.Various buffers may be used to achieve the desired pH and maintain thepH during the elution. Illustrative buffers include borate, phosphate,carbonate, tris, barbital and the like. The particular buffer employedis not critical, but in individual assays, one buffer may be preferredover another.

Moderate, and desirably substantially constant, temperatures arenormally employed for carrying out the assay. The temperatures for thechromotography and production of a detectable signal are generally inthe range of about 10°-50° C., more usually in the range of about15°-50° C., and frequently are ambient temperatures, that is, about15°-25° C.

The concentration of analyte which may be assayed generally varies fromabout 10⁻⁴ to about 10⁻¹⁵ M, more usually from about 10⁻⁶ to 10⁻¹⁴ M.Considerations, such as the concentration of the analyte of interest andthe protocol will normally determine the concentration of the otherreagents.

While the concentrations of many of the various reagents in the sampleand reagent solutions are generally determined by the concentrationrange of interest of the analyte, the final concentration of each of thereagents is normally determined empirically to optimize the sensitivityof the assay over the range of interest. However, with certainprotocols, individual reagents may be used in substantial excess withoutdetrimentally affecting the sensitivity of the assay.

Other reagents which are members of the signal producing system can varywidely in concentration depending upon the particular protocol and theirrole in signal production.

In carrying out the assay, the protocol normally involves dissolving thesample into the eluting solvent. The sample may be derived from a widevariety of sources, such as physiologic fluids, illustrated by blood,serum, plasma, urine, ocular lens fluid, spinal fluid, etc., chemicalprocessing streams, food, pesticides, pollutants, etc.

The bottom or proximal end of device 10 (i.e., the end of device 10(FIG. 1) that is contacted with the liquid medium) is contacted with thesample dispersed in the solvent, which is normally a buffered aqueousmedium which may contain one or more members of the signal producingsystem. Where a member of the signal producing system is present, atleast one member is usually conjugated to a sbp member to provide a sbpmember-label conjugate.

Sufficient time is allowed for the solvent front to complete traversalof piece of bibulous material 18.

For the most part, relatively short times are involved. Usually, thetraverse of the sample through first piece 18 takes at least 30 sec andnot more than 1 hour, more usually from about 1 min to 30 min.

A solutions that may include other reagents including signal producingsystem members not included with the sample solution or on piece 18 or20 are then contacted with device 10, and pressure is applied to arms 12and 14 to bring piece of bibulous material 22 into contact with piecesof bibulous material 18 and 20, respectively. This solution thenmigrates along piece of bibulous material 18 and subsequently alongpiece of bibulous material 20. After a suitable time the immunosorbingzone is examined for the presence of signal. The development of thesignal generally ranges from 30 sec to 30 min, more usually from about30 sec. to 5 min.

For quantitative assays, a standard sample can be provided having aknown amount of analyte. The analyte sample and the standard are eachrun on a different device 10, and a quantitative comparison is madebetween the standard sample and the analyte sample. If necessary, morethan one standard can be employed, so that the distance traversed forthe different concentrations can be used to quantitate a particularsample.

In carrying out an assay (see, for example, U.S. Ser. No. 928,771 filedNov. 7, 1986, the disclosure of which is incorporated herein byreference) utilizing an apparatus as depicted in FIGS. 5-9, the protocolwill normally involve combining in an aqueous medium the samplesuspected of containing the analyte with a first sbp member to form theaqueous test solution. The sample may be derived from a wide variety ofsources, such as physiologic fluids, illustrated by saliva, blood,serum, plasma, urine, ocular lens fluid, spinal fluid, etc., foodproducts such as milk and wine, chemical processing streams, food wastewater, etc.

A contact portion of first strip of bibulous material 82 (FIG. 5B),usually an end portion below the area containing zones 84, is contactedwith the test solution, usually by dipping of the contact portion intothe test solution. However, contact of the piece of bibulous materialwith the test solution can be carried out by other techniques such as byspotting the test solution on first piece of bibulous material 82.Wetting of the strip by capillary action usually is allowed to continue,for example, until the liquid front passes the last of zones 84.

For the most part, relatively short times are involved for the testsolution to traverse first strip 82. Usually, the traverse of the testsolution over strip 82 will take at least 30 sec and not more than 1hour, more usually from about 1 min to 30 min.

After the liquid has traversed first strip 82 at least past the last ofzones 84, enclosure 94 is removed from contact with any test solutionthat has not been absorbed. A liquid medium that can contain anyadditional chemical reagents that form part of the signal producingmeans that includes a label, is then placed in container 116 and housing106 is fitted into the top of 116. Next, enclosure 94 is inserted intohousing 106. The contact portion of the first strip 82 contacts theliquid medium. When flexible member 108 engages opening 102 in enclosure94, flexible member 108 contacts distortable portion 92 of second stripof bibulous material 90, forcing distortable portion 92 to contactportion 104 of first strip 82. As a consequence a capillary flowrelationship is created between strips 82 and 90 respectively.

After contacting the strip with the liquid medium, first strip 82 iscontacted with a developer solution containing any remaining members ofthe signal producing system not present in the sample solution or thesubsequent liquid medium, or present on strip 82. When an enzyme is usedas a label, a substrate will normally be in substantial excess in thedeveloper solution, so as not to be rate limiting (greater concentrationthan Km). The developer solution will usually be appropriately bufferedfor the enzyme system. A sufficient time is allowed to elapse prior tomeasuring the signal to allow liquid to fully wet first strip 82 and toproduce an amount of the signal producing compound required to definethe region of the zone in which the analyte or analytes are bound. Oncethe detectable signal has been produced, the presence or absence of theanalyte or analytes in the sample is known.

The strips can be coated with a wide variety of materials to provide forenhanced properties. Coatings may include protein coatings,polysaccharide coatings, synthetic polymers, sugars or the like, whichare used particularly to enhance the stability of the materialsconjugated to the strip. These compounds can also be used for improvedbinding of the materials, such as antibody binding or the like.

The strip, or the zones, can be activated with reactive functionalitiesto provide for covalent bonding of the organic materials to beconjugated to the strip such as those described in U.S. Pat. No.4,168,146, the relevant disclosure of which is incorporated herein byreference.

As mentioned above one embodiment of this invention can be employed toassay a sample suspected of containing one or more of a plurality ofanalytes such as drugs, thus screening for the presence of one or moreof such analytes. In this situation the test solution is formed bymixing together in an appropriate liquid medium the sample and aplurality of first sbp members, e.g., conjugates each comprising one ofthe analytes and a label. If it is only desired to know if any one ofthe drugs is present such as in a screening assay, first strip 82contains a single zone, such as would be employed for a single drug, towhich is bound a binding agent capable of specifically binding each ofthe first sbp members. If it is necessary to know which drugs arepresent, strip 82 contains a separate, preferably non-contiguous, zonefor each drug (as depicted in FIG. 5B). To each zone is bound a bindingagent such as a second sbp member capable of specifically binding to adifferent first sbp member. In either case it is necessary to havenon-diffusively and preferably homogeneously bound on the strip betweenthe zones and the contact portion a plurality of second sbp members eachrespectively capable of binding one of the above first sbp members.

As a result of the present invention the above assay protocols aregreatly simplified by eliminating or reducing the need for precisetiming and accurate volumetric dispensing.

Sbp members can be diffusively or non-diffusively bound to one or morepieces of bibulous material utilized in the present invention.

To enhance the versatility of the subject invention, the device orapparatus can be provided in packaged combination with a liquid mediumand other components in the same or separate containers as theinterreactivity of the components permit. For conducting an assay thekit can further include other separately packaged reagents forconducting an assay including members of the signal producing system,antibodies either labeled or unlabeled, supports, ancillary reagents,and so forth. Reagents can be provided so that the ratio of the reagentsprovides for substantial optimization of the method and assay.

Having described several embodiments of devices and methods of thepresent invention, by way of example and not limitation, it is to beunderstood that various changes in form and detail may be made thereinwithout departing from the scope and spirit of this invention or thescope of the appended claims.

What is claimed is:
 1. A method for creating a capillary flowrelationship among three pieces of bibulous material which prior toactuation are in a non-capillary flow relationship to each other whichcomprises:(a) providing a device which comprises (i) two pieces ofbibulous material each in a non-capillary flow relationship to theother, and (ii) a distortable member having affixed thereto a thirdpiece of bibulous material, said distortable member being disposed withrespect to said two pieces of bibulous material such that distortion ofsaid distortable member brings said third piece of bibulous materialinto contact with said two pieces of bibulous material, and (b)distorting said distortable member by insertion thereof into a containerhaving limited space resulting in constriction of said distortablemember, which thereby results in contact of said pieces of bibulousmaterial so as to achieve a capillary flow relationship among saidpieces.
 2. The method according to claim 1 wherein all three pieces ofbibulous material are paper strips.
 3. The method according to claim 1wherein said distortable member comprises two arms in a distortablerelationship to each other wherein one of said arms contains said twopieces of bibulous material and said other arm contains said third pieceof bibulous material.
 4. The method according to claim 3 wherein upondistortion of said arms, said third piece of bibulous material contactsboth of said two pieces of bibulous material thereby creating acapillary flow relationship among all three pieces.
 5. The methodaccording to claim 3 wherein upon distortion of said distortable member,said third piece of bibulous material affixed to said distortable membercontacts both of said two pieces of bibulous material attached to saidsupport thereby creating a capillary flow relationship among all threepieces.
 6. A kit for conducting an assay comprising in packagedcombination:(a) a device which permits longitudinal capillary flow of aliquid through three pieces of bibulous material which prior toactuation are in a non-capillary flow relationship to each other, whichdevice comprises:(i) two pieces of bibulous material each in anon-capillary flow relationship to the other and (ii) a distortablemember having affixed thereto a third piece of bibulous material, saiddistortable member being disposed with respect to said two pieces ofbibulous material such that distortion of said distortable member bringssaid third piece of bibulous material into contact with said two piecesof bibulous material, (b) a container adapted for insertion of saiddevice therein, wherein said container is adapted to actuate saiddevice, when said device is inserted into said container, by distortingsaid distortable member thereby bringing said third piece of bibulousmaterial into contact with said two pieces of bibulous material and (c)separately packaged reagents for conducting an assay, said reagentscomprising at least one member of a signal producing system.
 7. The kitaccording to claim 6 wherein at least one of said two pieces of bibulousmaterial has a member of a specific binding pair bound thereto.
 8. Adevice that permits capillary flow of a liquid through three pieces ofbibulous material which prior to actuation are in a non-capillary flowrelationship to each other which comprises:an element comprising (a) twopieces of bibulous material each in a non-capillary flow relationship tothe other, and (b) a distortable member having affixed thereto a thirdpiece of bibulous material and said two pieces of bibulous material,said distortable member being disposed with respect to said two piecesof bibulous material such that distortion of said distortable memberbrings said third piece of bibulous material into contact with said twopieces of bibulous material; anda container adapted for insertion ofsaid element therein, wherein said container is adapted to actuate saiddevice, when said element is inserted into said container, by distortingsaid distortable member thereby bringing said third piece of bibulousmaterial into contact with said two pieces of bibulous material.
 9. Thedevice according to claim 8 wherein when actuated by distorting, saiddistortable member initiates a capillary flow relationship among saidpieces.
 10. The device according to claim 8 wherein all three pieces ofbibulous material are paper strips.
 11. The device according to claim 8wherein said device contains two arms in a distortable relationship toeach other wherein one of said arms contains said two pieces of bibulousmaterial whereas said other arm comprises said distortable member andcontains said third piece of bibulous material.
 12. The device accordingto claim 11 wherein upon distortion of said arms, said third piece ofbibulous material contacts both of said pieces of bibulous materialthereby creating a capillary flow relationship among all three pieces.